The present invention relates to novel probes and to mixtures of such probes, in addition to the design, construction and use of such novel probes or mixtures thereof for detecting a target sequence of one or more mycobacteria, which probes are capable of detecting such organism(s) optionally present in a test sample, e.g. sputum, laryngeal swabs, gastric lavage, bronchial washings, biopsies, aspirates, expectorates, body fluids (spinal, pleural, pericardial, synovial, blood, pus, bone marrow), urine, tissue sections as well as food samples, soil, air and water samples and cultures thereof. The invention relates in particular to novel probes and mixtures thereof for detecting the presence of one or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC) and for detecting the presence of one or more mycobacteria other than mycobacteria of the Mycobacterium tuberculosis Complex (MOTT). The invention further relates to diagnostic kits comprising one or more of such probes. The probes of the present invention are surprisingly able to penetrate the cell wall of the mycobacteria, thus making possible the development of fast an easy-performed in situ protocols.
Tuberculosis is a very life-threatening and highly epidemic disease which is caused by infection with Mycobacterium tuberculosis. Tuberculosis is presently the predominant infectious cause of morbidity and mortality world-wide, and is estimated to kill about three million people annually. WHO estimates that the annual number of new cases of tuberculosis will increase from 7.5 million in 1990 to 10.2 million in 2000, an escalation that will result in approximately 90 million new cases during this decade. It is furthermore estimated that 30 million people will die from tuberculosis during the 1990s, which equals one quarter of preventable deaths among adults.
The prevalence of tuberculosis has been very high in the poorer parts of the world such as Asia, Africa and South-America, but in recent years an increase has also been observed in industrialised countries. This appears to be due to an interaction of various factors including i.a. patterns of migration, poorly organised tuberculosis programmes and nutrition problems. Furthermore, a serious threat will arise from the emergence of new strains that are drug resistant or worse, multi-drug resistant.
Mycobacteria are often divided into tuberculous mycobacteria, i.e. mycobacteria of the Mycobacterium tuberculosis Complex (MTC), and non-tuberculous mycobacteria, i.e. mycobacteria other than those of the Mycobacterium tuberculosis Complex (MOTT). The MTC group comprises apart from M. tuberculosis, M. bovis, M. africanum and M. microti. Mycobacteria of the MOTT group are not normally pathogenic to healthy individuals but may cause disease in immunocompromised individuals, e.g. individuals infected with HIV. Clinical relevant mycobacteria of the MOTT group are in particular M. avium, M. intracellulare, M. kansasii and M. gordonae, but also M. scrofulaceum, M. xenopi and M. fortuitum. 
M. avium and M. intracellulare together with M. paratuberculosis and M. lepraemurium constitute the Mycobacterium avium Complex (MAC). Extended with M. scrofulaceum, the group is named Mycobacterium avium-intracellulare-scrofulaceum Complex (MAIS).
It is well-known that treatment of mycobacterial infections with antibiotics may lead to the emergence of drug resistant strains. Many antibiotic drugs excert their effects by interfering with protein synthesis or with transcription. Studies of the molecular mechanisms underlying certain antibiotic resistance phenotypes in clinical mycobacterium isolates have revealed mutations in rRNA genes. The development of resistance because of mutation(s) located in the rRNA gene is likely to occur since slow-growing mycobacteria have only a single rRNA operon. All mycobacteria populations comprise a minority of drug resistant mutants that have arisen by spontaneous mutation. These mutated mycobacteria do normally not survive particularly well, but, when single-drug therapy is offered as treatment, the drug susceptible bacteria are killed, and only the resistant mutants will survive and multiply, and, thus at some point, constitute the majority of the mycobacteria population. The selection of drug resistant bacteria due to inadequate drug therapy leads to a state of so-called xe2x80x9cacquired drug-resistancexe2x80x9d. In contrast, xe2x80x9cprimary drug-resistancexe2x80x9d is used to characterise a situation where drug-resistant mycobacteria can be isolated from a patient who has never been treated for mycobacterial infection, and has infected with drug-resistant mycobacteria from an individual suffering from infection with an acquired drug resistant bacterium.
Today, drug-resistance is determined primarily phenotypically by culturing clinical samples, in which presence of mycobacteria have been demonstrated, in the presence of the individual drugs. This is unfortunately a very slow and time-consuming procedure as the result of the drug-resistance studies depends on the growth rate of the mycobacteria, which are well-known to be slow. Thus, the result is not available until after several weeks.
Although the incidence of drug-resistance is, at least not yet, very common, it is nevertheless very important that resistant strains are identified and eradicated. Therefore, it is of major importance to find a reliable and rapidly performed method of diagnosing drug-resistance.
Presently, the detection of mycobacteria by microscopy is the most prevalent method for diagnosis. The sample (e.g. an expectorate) is stained for the presence of acid-fast bacilli using e.g. Ziehl-Neelsen staining. However, staining for acid-last bacilli does not provide the necessary information about the type of infection, only whether acid fast bacilli are present in the sample, and this is in itself not sufficient information for establishing a diagnosis. Samples positive for acid fast bacilli may subsequently be cultured in order to be able to perform species identification.
Since Ziehl-Neelsen staining cannot be used to determine whether the infection is caused by mycobacteria of the MTC group or mycobacteria other than mycobacteria of the MTC group, a positive staining frequently leads to very costly isolation of all the patients with suspected M. tuberculosis infection as well as treatment with medicaments to which the patient may not even respond.
Since the sensitivity of acid fast staining is only approximately 104-105 per ml smear negative samples should also be cultured as culture-based tests are sensitive, and as it may be possible to detect 10-100 organisms per sample, but the result is not available before up to 8 weeks of culturing. Likewise, information about drug susceptibility is not available until after 1-3 weeks of further testing.
Different solid or liquid media (Loewenstein Jensen slants and Dubos broth) have traditionally been used for culturing of mycobacteria-containing samples. Newer media include ESP Myco Culture System (Difco), MB/BacT (Organon Teknika), BacTec (Becton Dickinson) and MGIT (Becton Dickinson). These test media are based on colourmetric or fluorometric detection of carbon dioxide or oxygen produced by mycobacterial metabolism, and adapted to automated systems for large scale testing.
Species identification is presently carried out following culturing using traditional biochemical methods or probe hybridisation assays (e.g. AccuProbe by Gen-Probe Inc, USA). There is, therefore, an increasing need for means allowing a more rapid distinction between mycobacteria of the MTC group and mycobacteria other than those of the MTC group, and for further species identification of those especially mycobacteria other than those of the MTC group.
A number of new attempts to replace the culture-based methods relies on molecular amplification technology. Several methods have emerged, among them the polymerase chain reaction (PCR), the ligase chain reaction and transcription mediated amplification. The basic principle of amplification methods is that a specific nucleic acid sequence of the mycobacteria is amplified to increase the copy number of the specific sequence to a level where the amplicon may be detectable. In principle, the methods offers the possibility of detecting only one target sequence, thus, in principle, making detection of mycobacteria present at low levels possible. However, it has become dear that the target amplification methods cannot replace culture-based methods as only samples which are positive by staining for acid fast bacilli (AFB) give a satisfactory sensitivity. Furthermore, specific problems exist for each method. The PCR method may give false negative results due to the presence of inhibitors such as hemoglobin. Another problem arises from cross-contamination of negative specimens and/or reagents with amplified nucleic acid present in the laboratory environment leading to false positive results. A disadvantage is that costly reagents are needed for performing these tests. Furthermore, specialised instrumentation is required, making these tests mainly useful in large specialised laboratories, and generally not applicable in smaller clinical laboratories.
Nucleic acid probes for detecting rRNA of mycobacteria have been described in for example U.S. Pat. No. 5,547,842, EP-A 0 572 120 and U.S. Pat. No. 5,422,242.
Considering the perspective and impact the disease has, the development of rapid and preferably easy-performed and further economic feasible diagnostic detection tests are of utmost importance and would be a very valuable tool in the fight against the spread of tuberculosis.
Peptide nucleic acids are pseudo-peptides with DNA-binding capability. The compounds were first reported in the early nineties in connection with a series of attempts to design nucleotide analogues capable of hybridising, in a sequence-specific fashion, to DNA and RNA, cf. WO 92/20702.
Hybridisation of peptide nucleic acid probes to DNA and to RNA has been shown to obey the Watson-Crick base pairing rules, and peptide nucleic acid probes have been found to hybridise to a DNA or a RNA target with higher affinity and specificity than the nucleic acid counterparts. These properties are ascribed to the uncharged, as opposed to the charged, structure of the peptide nucleic acid and DNA or RNA backbones, respectively, and to the high conformational flexibility of the peptide nucleic acid molecules. These featuresxe2x80x94together with the documented stability of peptide nucleic acid towards a variety of naturally occurring nucleases and proteases that usually degrade DNA, RNA or proteinsxe2x80x94invite for use of peptide nucleic acid probes as antisense therapeutic agents and opens potentially important applications in diagnostics.
The present invention relates to novel peptide nuclic acid probes and to mixtures of such probes for detecting a target sequence of one or more mycobacteria optionally present in a sample.
In a first aspect, the present invention relates to peptide nucleic acid probes for detecting a target sequence of one or more mycobacteria optionally present in a sample, said probes being capable of hybridising to a target sequence of mycobacterial rDNA, precursor rRNA or rRNA forming detectable hybrids. In another aspect, the invention relates to peptide nucleic acid probes, said probe being capable of hybridising to a target sequence of mycobacterial rDNA, precursor rRNA, or 23S, 16S or 5S rRNA forming detectable hybrids.
The peptide nucleic acid probes according to the present invention are capable of hybridising to a target sequence of mycobacterial rDNA, precursor rRNA, or 23S, 16S or 5S rRNA forming detectable hybrids, said target sequence being obtainable by
(a) comparing the nucleobase sequences of said mycobacterial rRNA or rDNA of one or more mycobacteria to be detected with the corresponding nucleobase sequence of organism(s), in particular other mycobacteria, from which said one or more mycobacteria are to be distinguished,
(b) selecting a target sequence of said rRNA or rDNA which includes at least one nucleobase differing from the corresponding nucleobase of the organism(s), in particular other mycobacteria, from which said one or more mycobacteria are to be distinguished, and
(c) determining the capability of said probe to hybridise to the selected target sequence to form detectable hybrids.
Furthermore, the peptide nucleic acid probes according to the invention are capable of hybridising to a target sequence of mycobacterial rDNA, precursor rRNA or 23S, 16S or 5S rRNA forming detectable hybrids, said probe being obtainable by
(a) comparing the nucleobase sequences of said mycobacterial rRRA or rDNA of one or more mycobacteria to be detected with the corresponding nucleobase sequence of organism(s), in particular other mycobacteria, in particular other mycobacteria, from which said one or more mycobacteria are to be distinguished,
(b) selecting a target sequence of said rRNA or rDNA which includes at least one nucleobase differing from the corresponding nucleobase of the organism(s), in particular other mycobacteria, from which said one or more mycobacteria are to be distinguished,
(c) synthesising said probe, and
(d) determining the capability of said probe to hybridise to the selected target sequence to form detectable hybrids.
In a further aspect, the invention relates to novel peptide nucleic acid probes for detecting a target sequence of one or more mycobacteria of the Mycobacteruim tuberculosis Complex (MTC), or for detecting a target sequence of one or more mycobacteria other than mycobacteria of the Mycobacterium tuberculosis Complex (MOTT) optionally present in a sample, which probes comprises from 6 to 30 polymerised peptide nucleic acid moieties, said probe being capable of hybridising to a target sequence of mycobacterial rDNA, precursor rRNA or 23S, 16S or 5S rRNA forming detectable hybrids. Suitable probes are those of formula (I) comprising from 10 to 30 polymerised moieties of formula (I) 
wherein each X and Y independently designate O or S,
each Z independently designates O, S, NR1, or C(R1)2, wherein each R1 independently designate H, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl,
each R2, R3 and R4 designate independently H, the side chain of a naturally occurring amino acid, the side chain of a non-naturally occurring amino acid, C1-4 alkyl, C1-4 alkenyl or C1-4 alkynyl, or a functional group, each Q independently designates a naturally occurring nucleobase, a non-naturally occurring nucleobase, an intercelator, a nucleobase-binding group, a label or H,
with the proviso that the probe comprising such subsequence is capable of forming detectable hybrids with the target sequence of said mycobacterial rDNA, precursor rRNA or 23S, 16S or 5S rRNA.
Suitable probes for detecting a target sequence of 23S rRNA of one or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC) optionally present in a sample comprise from 10 to 30 polymerised moieties of formula (I) as defined above, with the proviso that the Qs of adjacent moieties are selected so as to form a sequence of which a subsequence includes at least one nucleobase that is complementary to a nucleobase of M. tuberculosis 23S rRNA differing from the corresponding nucleobase of at least M. avium located within the following domains
Positions 149-158 in FIG. 1A,
Positions 220-221 in FIG. 1A,
Positions 328-361 in FIG. 1A and FIG. 1B,
Positions 453-455 in FIG. 1B,
Positions 490-501 in FIG. 1B,
Positions 637-660 in FIG. 1C,
Positions 706-712 in FIG. 1D,
Positions 762-789 in FIG. 1D,
Position 989 In FIG. 1D,
Positions 1068-1072 in FIG. 1D,
Position 1148 in FIG. 1E,
Positions 1311-1329 in FIG. 1E,
Positions 1361-1364 in FIG. 1F,
Position 1418 in FIG. 1F,
Positions 1563-1570 in FIG. 1F,
Positions 1627-1638 in FIG. 1G,
Positions 1675-1677 in FIG. 1G,
Position 1718 in FIG. 1G,
Positions 1734-1740 in FIG. 1H,
Positions 1967-1976 in FIG. 1H,
Positions 2403-2420 in FIG. 1H,
Positions 2457-2488 in FIG. 1I,
Positions 2952-2956 in FIG. 1I,
Positions 2966-2969 in FIG. 1J,
Positions 3000-3003 in FIG. 1J or
Positions 3097-3106 in FIG. 1J,
and further with the proviso that the probe comprising such subsequence is capable of forming detectable hybrids with a target sequence of said mycobacterial 23S rRNA.
Suitable probes for detecting a target sequence of 16S rRNA of one or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC) optionally present in a sample comprise from 10 to 30 polymerised moieties of formula (I) as defined above, with the proviso that the Qs of adjacent moieties are selected so as to form a sequence of which a subsequence includes at least one nucleobase that is complementary to a nucleobase of M. tuberculosis 16S rRNA differing from the corresponding nucleobase of at least M. avium located within the following domains
Positions 76-79 in FIG. 2A,
Positions 98-101 in FIG. 2A,
Positions 135-136 in FIG. 2A,
Positions 194-201 in FIG. 2B,
Positions 222-229 in FIG. 2B,
Position 242 in FIG. 2B,
Position 474 in FIG. 2C,
Positions 1136-1145 in FIG. 2C,
Positions 1271-1272 in FIG. 2C,
Positions 1287-1292 in FIG. 2D,
Position 1313 in FIG. 2D, or
Position 1334 in FIG. 2D,
and further with the proviso that the probe comprising such subsequence is capable of forming detectable hybrids with a target sequence of said mycobacterial 16S rRNA.
Suitable probes for detecting a target sequence of 5S rRNA of one or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC) optionally present in a sample comprise from 10 to 30 polymerised moieties of formula (I) as defined above, with the proviso that the Qs of adjacent moieties are selected so as to form a sequence of which a subsequence includes at least one nucleobase that is complementary to a nucleobase of M. tuberculosis 5S rRNA differing from the corresponding nucleobase of at least M. avium located within the following domain
Positions 86-90 in FIG. 3
and further with the proviso that the probe comprising such subsequence is capable of forming detectable hybrids with a target sequence of said mycobacterial 5S rRNA.
In a preferred aspect, the invention relates to peptide nucleic acid probes for detecting a target sequence of 23S or 16S rRNA of one or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC) optionally present in a sample comprising from 10 to 30 polymerised moieties of formula (I) as defined above, with the proviso that the Qs of adjacent moieties are selected so as to form a sequence of which a subsequence includes at least one nucleobase that is complementary to a nucleobase of M. tuberculosis 23S or 16S rRNA differing from the corresponding nucleobase of at least M. avium located within the following domains
Positions 149-158 in FIG. 1A,
Positions 328-361 in FIG. 1A and FIG. 1B,
Positions 490-501 in FIG. 1B,
Positions 637-660 in FIG. 1C,
Positions 762-789 in FIG. 1D,
Positions 1068-1072 in FIG. 1D,
Positions 1311-1329 in FIG. 1E,
Positions 1361-1364 in FIG. 1F,
Positions 1563-1570 in FIG. 1F,
Positions 1627-1638 in FIG. 1G,
Positions 1734-1740 in FIG. 1H,
Positions 2457-2488 in FIG. 1I,
Positions 2952-2956 in FIG. 1I,
Positions 3097-3106 in FIG. 1J,
Positions 135-136 in FIG. 2A, or
Positions 1287-1292 in FIG. 2D,
and further with the proviso that the probe comprising such subsequence is capable of forming detectable hybrids with a target sequence of said mycobacterial 23S or 16S rRNA.
In a further embodiment, the present invention relates to peptide nucleic acid probes for detecting a target sequence of 23S rRNA of one or more mycobacteria other than mycobacteria of the Mycobacterium tuberculosis Complex (MOTT) optionally present in a sample comprising from 10 to 30 polymerised moieties of formula (I) as defined above, with the proviso that the Qs of adjacent moieties are selected so as to form a sequence of which a subsequence includes at least one nucleobase that is complementary to a nucleobase of M. avium 23S rRNA differing from the corresponding nucleobase of at least M. tuberculosis located within the following domains
Positions 99-101 in FIG. 4A,
Position 183 in FIG. 4A,
Positions 261-271 in FIG. 4A,
Positions 281-284 in FIG. 4B,
Positions 290-293 in FIG. 4B,
Positions 327-335 in FIG. 4B,
Positions 343-357 in FIG. 4B,
Positions 400-405 in FIG. 4B and FIG. 4C,
Positions 453-462 in FIG. 4C,
Positions 587-599 in FIG. 4C,
Positions 637-660 in FIG. 4D,
Positions 704-712 in FIG. 4D,
Positions 763-789 in FIG. 4E,
Positions 1060-1074 in FIG. 4E,
Positions 1177-1185 in FIG. 4E,
Positions 1259-1265 in FIG. 4F,
Positions 1311-1327 in FIG. 4F,
Positions 1345-1348 in FIG. 4F,
Positions 1361-1364 in FIG. 4G,
Positions 1556-1570 in FIG. 4G,
Positions 1608-1613 in FIG. 4H,
Positions 1626-1638 in FIG. 4H,
Positions 1651-1659 in FIG. 4H,
Positions 1675-1677 in FIG. 4H,
Positions 1734-1741 in FIG. 4H,
Positions 1847-1853 in FIG. 4I,
Positions 1967-1976 in FIG. 4I,
Positions 2006-2010 in FIG. 4I,
Positions 2025-2027 in FIG. 4I,
Positions 2131-2132 in FIG. 4J,
Positions 2252-2255 in FIG. 4J,
Positions 2396-2405 in FIG. 4J and FIG. 4K,
Positions 2416-2420 in FIG. 4K,
Positions 2474-2478 in FIG. 4K,
Position 2687 in FIG. 4K,
Position 2719 in FIG. 4K,
Position 2809 in FIG. 4L,
Positions 3062-2068 in FIG. 4L, or
Positions 3097-3106 in FIG. 4L,
and further with the proviso that the probe comprising such subsequence is capable of forming detectable hybrids with a target sequence of said mycobacterial 23S rRNA.
The invention further relates to peptide nucleic acid probes for detecting a target sequence of 16S rRNA of one or more mycobacteria other than mycobacteria of the Mycobacterium tuberculosis Complex (MOTT) optionally present in a sample comprising from 10 to 30 polymerised moieties of formula (I) as defined above, with the proviso that the Qs of adjacent moieties are selected so as to form a sequence of which a subsequence includes at least one nucleobase that is complementary to a nucleobase of M. avium 16S rRNA differing from the corresponding nucleobase of at least M. tuberculosis located within the following domains
Positions 135-136 in FIG. 5A,
Positions 472-475 in FIG. 5A,
Positions 1136-1144 in FIG. 5A,
Positions 1287-1292 in FIG. 5B,
Position 1313 in FIG. 5B, or
Position 1334 in FIG. 5B,
and further with the proviso that the probe comprising such subsequence is capable of forming detectable hybrids with a target sequence of said mycobacterial 16S rRNA.
In a preferred embodiment, the invention relates to peptide nucleic acid probes for detecting a target sequence of 23S or 16S rRNA of one or more mycobacteria other than mycobacteria of the Mycobacterium tuberculosis Complex (MOTT) optionally present in a sample, which probes comprise from 10 to 30 polymerised moieties of formula (I) as defined above, with the proviso that the Qs of adjacent moieties are selected so as to form a sequence of which a subsequence includes at least one nucleobase that is complementary to a nucleobase of M. avium 23S or 16S rRNA differing from the corresponding nucleobase of at least M. tuberculosis located within the following domains
Positions 99-101 in FIG. 4A,
Positions 290-293 in FIG. 4B,
Positions 400-405 in FIG. 4B and FIG. 4C,
Positions 453-462 in FIG. 4C,
Positions 637-660 in FIG. 4D,
Positions 763-789 in FIG. 4E,
Positions 1311-1327 in FIG. 4F,
Positions 1361-1364 in FIG. 4G,
Positions 1734-1741 in FIG. 4H,
Positions 2025-2027 in FIG. 4I,
Positions 2474-2478 in FIG. 4K,
Positions 3062-2068 in FIG. 4L, or
Positions 1287-1292 in FIG. 5B,
and further with the proviso that the probe comprising such subsequence is capable of forming detectable hybrids with a target sequence of said mycobacterial 23S or 16S rRNA.
In another embodiment, the present invention relates to peptide nucleic acid probes for detecting a target sequence of 23S, 16S or 5S rRNA of one or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC) or for detecting a target sequence of 23S, 16S or 5S rRNA of one or more mycobacteria other than mycobacteria of the Mycobacterium tuberculosis Complex (MOTT), in particular drug resistant mycobacteria, optionally present in a sample, which probes comprise from 10 to 30 polymerised moieties of formula (I) as defined above, with the proviso that the Qs of adjacent moieties are selected so as to form a sequence of which a subsequence includes at least one nucleobase that is complementary to a nucleobase that differs from the corresponding nucleobase of 23S, 16S or 5S rRNA of said one or more mycobacteria located within the following domains
Positions 2568-2569 in FIG. 6,
Position 452 in FIG. 7,
Positions 473-477 in FIG. 7, or
Positions 865-866 in FIG. 7,
and further with the proviso that the probe comprising such subsequence is capable of forming detectable hybrids with the target sequence of said mycobacterial 23S, 16S or 5S rRNA.
In preferred embodiments, the peptide nucleic acid probes according to the invention are those of formula (II), (III), or (IV) 
wherein Z, R2, R3, and R4, and Q is as defined above, and further with the provisos defined above. In may especially be preferred that Z is NH, NCH3 or O, each R2, R3 and R4 independently designate H or the side chain of a naturally occurring amino acid, the side chain of a non-naturally occurring amino acid, or C1-4 alkyl, and each Q is a naturally occurring nucleobase or a non-naturally occurring nucleobase. In a further preferred embodiment, Z is NH or O, and R2 is H or the side chain of Ala, Asp, Cys, Glu, His, HomoCys, Lys, Orn, Ser or Thr, and Q is a nucleobase selected from thymine, adenine, cytosine, guanine, uracil, iso-C and 2,6-diaminopurine. The peptide nucleic acid probes may suitably be those of formula (V) 
wherein R4 is H or the side chain of Ala, Asp, Cys, Glu, His, HomoCys, Lys, Orn, Ser or Thr, and Q is as defined above, and with the provisos defined above.
Such peptide nucleic acid probes may further comprise one or more labels and a mixture of such probes, which labels may be mutually identical or different, which probes optionally may comprise one or more linkers, and which probes may be mutually identical or different with the provisos defined above.
For many applications, it is preferred that the nucleobase sequence of the peptide nucleic acid probes is substantially complementary to the nucleobase sequence of the target sequence. In preferred embodiments, the nucleobase sequence of said probe is complementary to the nucleobase sequence of said target sequence.
Peptide nucleic acid probes for detecting a target sequence of one or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC) or for deflecting a target sequence of one or more mycobacteria other than mycobacteria of the Mycobacterium tuberculosis Complex (MOTT) are suitably those wherein the Qs of adjacent moieties are selected so as to form the following subsequences
In a preferred embodiment, such probes are those wherein the Qs of adjacent moieties are selected so as to form the following subsequences
In accordance herewith, the present invention relates to peptide nucleic acid probes selected from
wherein Flu denotes a 5-(and 6)-carboxyfluoroescein label and Rho denotes a rhodamine label.
In a further aspect, the invention relates to the use of peptide nucleic acid probes as defined above or a mixture thereof for detecting a target sequence of one or more mycobacteria optionally present in a sample. In particular, the invention relates to the use of a peptide nucleic acid probe or a mixture thereof for detecting a target sequence of one or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC), in particular a target sequence of M. tuberculosis, and further to the use of peptide nucleic acid probes or a mixture thereof for detecting a target sequence of one or more mycobacteria other than mycobacteria of the Mycobacterium tuberculosis Complex (MOTT), in particular a target sequence of one or more mycobacteria of the Mycobacterium avium Complex.
The invention further relates to a method for detecting a target sequence of one or more mycobacteria optionally present in a sample comprising
(1) contacting any rRNA or rDNA present in said sample with one or more peptide nucleic acid probes as defined above or a mixture thereof under conditions, whereby hybridisation takes place between said probe(s) and said rRNA or rDNA, and
(2) observing or measuring any formed detectable hybrids, and relating said observation or measurement to the presence of a target sequence of one or more mycobacteria in said sample.
In particular, the invention relates to a method for detecting a target sequence of one or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC), in particular a target sequence of M. tuberculosis, or to a method for detecting a target sequence of one or more mycobacteria other than mycobacteria of the Mycobacterium tuberculosis Complex (MOTT). In preferred embodiments, the hybridisation takes place in situ, or takes place in vitro. In an embodiment, a signal amplifying system is used for measuring the resulting hybridisation. It is further preferred that the sample is a sputum sample.
Furthermore, the invention relates to kits for detecting a target sequence of one or more mycobacteria, in particular a target sequence of one or more mycobacteria of the Mycobacterium tuberculosis Complex (MTC), and in particular a target sequence of M. tuberculosis, and/or for detecting a target sequence of one or more mycobacteria other than mycobacteria of the Mycobacterium tuberculosis Complex (MOTT), in particular a target sequence of one or more mycobacteria of the Mycobacterium avium Complex (MAC), which kit comprise at least one peptide nucleic acid probe as defined above, and optionally a detection system with at least one detecting reagent in one embodiment thereof, the kit further comprises a solid phase capture system.